Squamous cell carcinoma, a cancer which afflicts sites including the gastrointestinal and airway mucosa, is by far the predominant form of oral cancer. Paradoxically, while oral squamous cell carcinoma, OSCC, typically initiates as visible lesions in the oral cavity, early detection of the disease at this curable stage is limited. This is likely due to the high number of other more innocuous oral lesions that occur that can resemble OSCC, and the necessity for a surgical biopsy for accurate detection and diagnosis of the disease. Brush oral cytology can be used to collect cells from suspect lesions without surgery, which in turn can be used for microscopic analysis of malignant changes but with decreased accuracy. We propose a method of lesion evaluation that uses this same noninvasive procedure, brush cytology, but to provide RNA to allow gene expression analysis of the lesion mucosa. We have shown that this methodology, contrary to preconceived ideas that it would not be feasible, provided high quality RNA capable of allowing the differentiation of OSCC from normal mucosa in a hamster oral cancer model. We will take advantage of recent studies that have shown in surgically biopsied tissue that class predictors, based on changes in global gene expression, can be developed that can accurately diagnose malignant disease. The study proposed will determine if RNA from cytology can be analyzed to produce a global gene expression signature that can be used to detect OSCC. Prospective OSCC patients, prior to undergoing standard biopsy diagnosis for OSCC, will be subjected to brush oral cytology to allow us to develop, and then validate, a gene expression signature that identifies OSCC. An immediate result will be a noninvasive methodology to accurately detect and contribute to the diagnosis of OSCC, ready to be further validated in other clinics. Next, we will apply this same approach to develop a gene expression based class predictor for the earliest stage of squamous cell carcinoma, moderate to severe dysplasia and carcinoma in situ. After analysis of RNA from brush cytology from pre-cancerous oral lesions, we will use statistical methodologies optimized for heterogeneous samples to develop a class predictor for these early malignancies based on gene expression of a small set of genes. In the end, we will start to determine if the detection and diagnosis of oral premalignancies and OSCC using RNA from brush cytology is superior to the OralCDx system. 1 PUBLIC HEALTH RELEVANCE: These results should deliver a noninvasive method for detection of the vast majority of oral cancers that is validated against even the earliest stages of the disease. It will be ready to be tested in additional clinics with other investigators. This should allow the earlier detection of oral cancer and could save lives immediately. This finding would strongly support the usage of the RNA from brush cytology approach with other gastrointestinal and airway passage cancers. This study would also determine the feasibility of noninvasive brush cytology methods to detect oral premalignancies and deliver a prototype predictor. With some modifications, such as the usage of commercially available room temperature RNA stabilization solutions, this would bring us much closer to an economical, noninvasive form of oral cancer screening.